CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qian, W.
Right arrow Articles by Wiman, K. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qian, W.
Right arrow Articles by Wiman, K. G.
Cell Growth & Differentiation Vol. 11, 31-39, January 2000
© 2000 American Association for Cancer Research

Articles

Polyoma Virus Middle T and Small t Antigens Cooperate to Antagonize p53-induced Cell Cycle Arrest and Apoptosis1

Wang Qian and Klas G. Wiman2

Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden

Abstract

Wild-type p53 triggers two distinct biological responses, cell cycle arrest and apoptosis. Several small DNA tumor viruses encode proteins that bind p53 and thus block the function of p53. This probably reflects the need of these viruses to prevent p53-induced cell cycle arrest and apoptosis to allow viral DNA replication. Unlike SV40 large T, polyoma virus large T does not bind p53, and it is still unclear how polyoma virus blocks p53 function. To address this question, we transfected polyoma virus middle T or small t alone or middle T and small t together into J3D mouse T-lymphoma cells carrying temperature-sensitive p53 (ts p53). Induction of wild-type p53 by temperature shift to 32°C triggered both G1 cell cycle arrest and apoptosis in parental J3D-ts p53 cells. In contrast, J3D-ts p53 cells coexpressing middle T and small t showed only a weak G1 cell cycle arrest response after induction of wild-type p53 at 32°C. Fluorescence-activated cell sorter analysis revealed that nearly half of the middle T-expressing cells, 30% of the small t-expressing cells, and a majority of the cells coexpressing middle T and small t were resistant to p53-induced apoptosis. The phosphatidylinositol 3-kinase inhibitor wortmannin partially abrogated the protective effect of middle T but not small t on p53-induced apoptosis, indicating that middle T prevents p53-induced apoptosis through the phosphatidylinositol 3-kinase signal transduction pathway. Our results thus establish a mechanism for polyoma virus-mediated inhibition of p53 function.

Introduction

The p53 tumor suppressor gene is frequently mutated in human and rodent tumors (1) , indicating that p53 plays a critical role in the defense against tumor development. This notion is further supported by the observation that p53 knock-out mice are highly susceptible to spontaneous tumors (2) . Accumulation of wild-type p53 in response to various forms of cellular stress, including DNA damage, oncogene activation, and hypoxia, can trigger both cell cycle arrest and apoptosis (reviewed in Refs. 3 and 4 ). Loss of wild-type p53 function may thus allow survival and growth of emerging tumor cells driven to proliferate by activated oncogenes and/or loss of normal G1 cell cycle control and tumor growth under hypoxic conditions. p53-induced apoptosis plays an important role for p53-mediated tumor suppression in vivo (5) .

p53 is a transcription factor that activates expression of target genes containing p53 DNA binding sites. Target genes involved in p53-induced cell cycle arrest include p21 (6 , 7) , GADD45 (8) , 14-3-3 {varsigma} (9) , and possibly B99 (10) , whereas Bax (11) , Fas (12) , KILLER/DR5 (13) , IGF-BP3 (14) , and the PIG (15) genes have been implicated in p53-induced apoptosis (16 , 17) . p53 mutations in tumors are mostly missense mutations that cause single amino acid substitutions in the specific DNA-binding core domain of p53 (3) , suggesting that specific DNA binding, and thus transactivation of target genes, is crucial for p53-mediated tumor suppression. p53 can also be inactivated at the protein level through interactions with DNA viral oncoproteins (18 , 19) . The adenovirus E1B 55K protein binds to the NH2 terminal domain of p53 and blocks its transcriptional transactivation function. The adenovirus E1B 19K protein, which is a functional homologue of the antiapoptotic Bcl-2 protein, prevents p53-induced apoptosis through interactions with several cellular proteins, including Bax and Bak. The SV40 large T antigen inhibits sequence-specific DNA binding of p53 by interacting with the p53 core domain, and the E6 proteins encoded by human papillomaviruses can bind p53 and catalyze ubiquitin-mediated degradation of p53. The same three viruses also encode oncoproteins that interact with pRb,3 another key regulator of cell growth and survival (20) . These viral oncoproteins have presumably evolved to ensure cell survival and viral DNA replication after virus infection and thus production of viral progeny.

Py is a small DNA tumor virus that induces various tumors in newborn mice, rats, hamsters, and rabbits (21) . Its early region encodes three proteins: the large T (PyLT), middle T (PymT), and small T (Pyst) antigens, with molecular weights of Mr 100,000, Mr 55,000, and Mr 22,000, respectively (22) . The SV40 and PyLT antigens share a high degree of sequence identity as well as functional and biochemical characteristics (23 , 24) . Unlike SV40 large T, however, PyLT does not bind p53 (25) . There is no evidence indicating that the PymT or Pyst antigens can complex with p53. This raises the question as to how the Py interferes with p53-induced cell cycle arrest and apoptosis.

To investigate whether PymT and/or Pyst may interfere with p53 function, we expressed PymT or Pyst alone or PymT in combination with Pyst in J3D mouse T-lymphoma cells carrying ts p53. Induction of wild-type p53 by temperature shift to 32°C triggers cell cycle arrest and apoptosis in these cells (26 , 27) . We show here that coexpression of PymT and Pyst inhibits both p53-induced cell cycle arrest and apoptosis.

Results

PymT Overexpression Impairs p53-induced Apoptosis.
J3D-ts p53 cells (clone J3DM6H4) were stably transfected with the PymT expression vector pBabe-mT by electroporation and cloned by limiting dilution. The presence of the pBabe-mT construct in individual clones was examined by PCR using PymT-specific primers (data not shown). PymT-carrying clones were further examined for the expression of mT protein by Western blot analysis using the rat monoclonal anti-PymT antibody 815 (Fig. 1)Citation . To confirm that the PymT-positive clones had retained expression of ts p53, cells were also analyzed by Western blotting using the p53-specific monoclonal antibody PAb122 (Fig. 1)Citation . Clones expressing both ts p53 and PymT were selected for further analysis.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1. Western blot analysis of the expression of p53 and PymT in PymT-transfected M6H4 cells using the anti-p53 monoclonal antibody PAb122 and the anti-PymT monoclonal antibody 815. Left (in thousands), molecular weight markers. J3D, the parental p53-negative mouse T lymphoma line; M6H4, a clone of J3D transfected with mouse ts p53; vector, M6H4 cells transfected with the empty vector pBabe-puro; MT pool, pool of M6H4 cells transfected with PymT; MT1–10, clones of PymT-transfected M6H4 cells.

 
To examine the effect of PymT on wild-type p53-induced cell cycle arrest, pooled transfected cells and 10 individual clones of PymT/ts p53-expressing cells were analyzed for cell cycle distribution by FACS after induction of wild-type p53 by temperature shift from 37°C to 32°C for 24 h. Parental J3D-ts p53 cells (J3DM6H4) and parental cells transfected with empty vector were used as controls. No major effect of PymT on p53-induced cell cycle arrest was observed in the pooled transfected cells nor in any of the clones. Representative examples are shown in Fig. 2Citation and Table 1Citation . To examine whether PymT affects p53-induced apoptosis, the viability of pooled transfectants and 10 clones expressing PymT and ts p53 were analyzed by propidium iodide staining and FACS in the absence of detergent (28) at 48 h after temperature shift to 32°C. At least 90% of the parental J3DM6H4 cells or the parental cells transfected with empty vector were apoptotic after 48 h at 32°C. In contrast, only ~24% of the pooled PymT-transfected cells were dead at this time point (Figs. 3Citation and 4)Citation . The different PymT-transfected clones also showed increased resistance to p53-induced apoptosis. The fraction of dead cells varied between 29 and 81% among these clones, with an average of 54% (Figs. 3Citation and 4Citation ; data not shown). The ability of PymT to prevent p53-induced death is also demonstrated by the decreased fraction of PymT-expressing cells with sub-G1 DNA content in Fig. 2Citation (see also Table 1Citation ). Thus, PymT alone can at least partially block wild-type p53-triggered apoptosis.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2. Cell cycle analysis of PymT-expressing M6H4 cells. Logarithmically growing cells were seeded and harvested after incubation for 24 h at 37°C or 32°C and analyzed by FACS. The positions of cells with 2N (G0-G1) and 4N (G2-M) DNA content are indicated. Similar results were obtained with other PymT-expressing M6H4 clones (not shown). Parental and vector-transfected M6H4 cells were used as controls.

 

View this table:
[in this window]
[in a new window]
  		Table 1 Cell cycle distribution

The percentage of cells in different phases of the cell cycle is indicated.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. Analysis of p53-induced apoptosis by propidium iodide staining and FACS. Pooled PymT-expressing cells or clones thereof were harvested after incubation for 48 h at the indicated temperatures. X axis, relative fluorescence intensity, which is proportional to plasma membrane permeability. Viable (M1) and dead (M2) cells are indicated. Similar results were obtained with other PymT-expressing clones (not shown).

 


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 4. Diagram showing the fraction of apoptotic cells among the pooled or cloned cells expressing PymT, PymT+st, or Pyst 48 h after temperature shift to 32°C, as determined by FACS analysis. Parental and vector-transfected M6H4 cells were used as controls.

 
Coexpression of PymT and Pyst Inhibits p53-triggered Cell Cycle Arrest and Apoptosis.
Because transformation by PymT may require cooperation with either PyLT or Pyst (29, 30, 31) , we asked whether PymT and Pyst were able to cooperate to block p53-induced cell cycle arrest and/or apoptosis. PymT and Pyst were transfected together into J3DM6H4 cells, and clones expressing both proteins were identified by Western blot analysis using the 815 monoclonal antibody specific for PymT and the F4 monoclonal antibody that detects all three polyoma T antigens. Expression of ts p53 was confirmed using PAb122 (Fig. 5)Citation . Cell cycle distribution and apoptosis in pooled and individual PymT+st transfectants, parental cells, and empty vector transfectants were analyzed by FACS as described above.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 5. Western blot analysis of the expression of p53, PymT, and Pyst in PymT+st-transfected M6H4 cells using the anti-p53 monoclonal antibody PAb122, the anti-PymT monoclonal antibody 815, and the monoclonal antibody F4 that detects all three Py T antigens. Left (in thousands), molecular weight markers. J3D, the parental p53-negative mouse T lymphoma line; M6H4, a clone of J3D transfected with mouse ts p53; vector, M6H4 cells transfected with the empty vector pBabe-puro; M+S pool, pool of M6H4 cells transfected with PymT+st; M+S1–10, clones of PymT+st-transfected M6H4 cells.

 
Whereas a majority of the parental J3DM6H4 cells and the vector transfectants were arrested in G1 24 h after induction of wild-type p53 by temperature shift to 32°C, the PymT+st-expressing cells (pooled transfectants and 10 individual clones) showed only a minor increase in the number of cells in G1 and a less pronounced reduction in the number of cells in S phase compared with the control cells at the same time point (Fig. 6Citation and Table 1Citation ; data not shown). Moreover, FACS analysis of cell survival at 48 h after temperature shift revealed that a major fraction of the PymT+st-expressing cells were resistant to p53-induced apoptosis (Fig. 7Citation ; summarized in Fig. 4Citation ). Only between 12 and 52% (average, 26%) of the pooled transfectants and individual clones were apoptotic at this time, whereas 90% or more of the parental cells and the parental cells transfected with empty vector had died by apoptosis. The PymT+st transfectants survived and continued to proliferate at least 4 days after temperature shift to 32°C (data not shown). The antiapoptotic effect of PymT+st is also evident from the reduction in the number of sub-G1 cells in Fig. 6Citation and Table 1Citation . These results demonstrate that coexpression of PymT and Pyst can inhibit both p53-induced cell cycle arrest and apoptosis.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 6. Cell cycle analysis of PymT+st-expressing M6H4 cells. Logarithmically growing cells were seeded and harvested after incubation for 24 h at 37°C or 32°C and analyzed by FACS. The positions of cells with 2N (G0-G1) and 4N (G2-M) DNA content are indicated. Similar results were obtained with other PymT+st-expressing M6H4 clones (not shown). Parental and vector-transfected M6H4 cells were used as controls.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 7. Analysis of p53-induced apoptosis by propidium iodide staining and FACS. Pooled PymT+st-expressing cells or clones thereof were harvested after incubation for 48 h at the indicated temperatures. Viable (M1) and dead (M2) cells are indicated. Similar results were obtained with other PymT+st-expressing clones (not shown).

 
To determine whether PymT and/or Pyst were able to prevent apoptosis induced by agents or conditions other than p53 activation, parental M6H4 cells and cells expressing one or both Py proteins were grown in medium containing 0.1% FCS. At least 50% of the cells in all cultures underwent apoptosis within 3 days, regardless of PymT/Pyst expression (data not shown). Therefore, neither PymT or Pyst alone nor coexpression of PymT+st protects these cells from serum starvation-induced apoptotic death.

Pyst Alone Partially Inhibits p53-induced Apoptosis.
Because our results showed that Pyst could cooperate with PymT to block p53-induced growth arrest and apoptosis, we asked whether Pyst alone could interfere with these two distinct functions of p53. Pyst was transfected into J3DM6H4 cells, and expression of both Pyst and p53 in pooled transfectants was confirmed by Western blot with the monoclonal antibodies F4 and PAb122, respectively (Fig. 8)Citation . Cell cycle distribution and apoptosis in pooled transfectants, parental cells, and empty vector transfectants were analyzed by FACS as described above. No effect of Pyst on p53-induced cell cycle arrest was observed at 22.5 h after temperature shift from 37°C to 32°C (Fig. 9Citation ; Table 1Citation ). However, ~30% of the Pyst transfectants remained viable at 48 h after temperature shift to 32°C (Fig. 10)Citation . Thus, Pyst alone inhibited p53-induced apoptosis but did not completely block it.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 8. Western blot analysis of the expression of p53 and Pyst in Pyst-transfected M6H4 cells using the monoclonal antibodies 122 and F4. Left (in thousands), molecular weight markers. J3D, the parental p53-negative mouse T lymphoma line; M6H4, a clone of J3D transfected with mouse ts p53; vector, M6H4 cells transfected with the empty vector pBabe-puro; ST pool, pool of M6H4 cells transfected with Pyst.

 


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 9. Cell cycle analysis of Pyst-expressing M6H4 cells. Logarithmically growing cells were seeded and harvested after incubation for 22.5 h at 37°C or 32°C and analyzed by FACS. The positions of cells with 2N (G0-G1) and 4N (G2-M) DNA content are indicated. Parental and vector-transfected M6H4 cells were used as controls.

 


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 10. Analysis of p53-induced apoptosis by propidium iodide staining and FACS. Pooled Pyst-expressing cells were harvested after incubation for 48 h at the indicated temperatures. Viable (M1) and dead (M2) cells are indicated. Parental and vector-transfected M6H4 cells were used as controls.

 
Inhibition of p53-triggered Cell Cycle Arrest and Apoptosis by PymT+st Coexpression Is Independent of p21 and Bax Transactivation.
To test whether the reduction in p53-mediated cell cycle arrest and apoptosis in cells coexpressing PymT and Pyst is associated with a decreased expression of the p53 target genes p21 and Bax, the pooled PymT+st-transfected cells and empty vector-transfected cells were treated with Wortmannin after temperature shift to 32°C and analyzed by Northern blotting. Both p21 and Bax were induced to similar levels at 3 h after temperature shift to 32°C in the presence or absence of wortmannin in pooled PymT+st or vector-transfected control cells (data not shown). This demonstrates that PymT+st coexpression does not inhibit p53-induced cell cycle arrest and apoptosis through disruption of p53-mediated transactivation of p21 and Bax.

PymT-mediated Inhibition of p53-induced Apoptosis Involves the PI3 Kinase Signal Transduction Pathway.
The PI3 kinase signaling pathway can transduce a survival signal (32 , 33) . PymT can form a complex with the Mr 85,000 regulatory subunit of PI3 kinase and accelerate activation of PI3 kinase, which is one of the mechanisms for PymT-mediated transformation (34, 35, 36, 37) . To determine whether PymT and/or Pyst inhibited p53-induced apoptosis through the PI3 kinase pathway, we treated PymT, Pyst, or PymT+st transfectants with the PI3 kinase inhibitor wortmannin (38) . After incubation of cells for 15 h at 37°C or 32°C, wortmannin was added at 3-h intervals for 11 h at a final concentration of 1 µM. Cell survival was then examined by propidium iodide staining and FACS analysis. Wortmannin had no effect on cell viability at 37°C (not shown). At 32°C, however, addition of wortmannin resulted in a significant increase in p53-induced apoptosis in the pooled PymT and PymT+st-transfectants but not in the pooled Pyst transfectants. As shown in Fig. 11Citation , the fraction of apoptotic PymT-expressing cells increased from 24 to 48%, and the fraction of apoptotic PymT+st-expressing cells increased from 9 to 28%, in the presence of wortmannin. In contrast, wortmannin treatment caused only a minor increase in p53-induced apoptosis in the vector-transfected J3DM6H4 control cells and pooled Pyst transfectants. These results suggest that PymT and PymT+st block p53-induced apoptosis at least partially through the PI3 kinase signal transduction pathway, whereas the effect of Pyst is PI3 kinase independent.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 11. Effect of wortmannin on PymT, Pyst, or PymT+st-mediated protection from p53-induced apoptosis. Logarithmically growing cells were seeded at 2 x 105/ml and incubated for 15 h at 37°C or 32°C. Wortmannin was then added at 3-h intervals for 11 h at a final concentration of 1 µM during continued incubation at the same temperature. p53-induced apoptosis was analyzed by propidium iodide staining and FACS.

 
In addition, we tested the effect of wortmannin on the cooperative inhibition of p53-induced cell cycle arrest by PymT and Pyst. Wortmannin treatment partially restored p53-induced G1 cell cycle arrest in PymT+st transfectants, as evidenced by an increased fraction of cells with G1 DNA content and a decreased fraction of cells in the S and G2-M phases (data not shown). This indicates that PymT+st-mediated protection against p53-induced cell cycle arrest also involves the PI3 kinase pathway.

Discussion

The fact that the PyLT antigen does not complex with p53 led us to ask whether Py is able to interfere with p53 function in a more indirect way. To study possible functional interactions between p53 and the PymT and Pyst antigens, we expressed PymT and/or Pyst in J3D-ts p53 cells in which induction of wild-type p53 by temperature shift to 32°C triggers both cell cycle arrest and apoptosis (26 , 27) . We found that PymT or Pyst alone did not significantly affect p53-induced cell cycle arrest but partially impaired p53-induced apoptosis. However, coexpression of PymT and Pyst had a marked inhibitory effect on both p53-dependent biological responses, at least partially abrogating p53-induced cell cycle arrest and strongly inhibiting p53-induced apoptosis. PymT and Pyst may provide a prolonged or perhaps permanent protective effect, because PymT+st transfectants were alive and proliferated exponentially at least 4 days after activation of wild-type p53 (data not shown). These findings thus suggest that Py, which does not encode any protein that physically interacts with p53, can nonetheless prevent p53-induced cell cycle arrest and apoptosis in an indirect manner through the PymT and Pyst proteins. This resolves the apparent paradox that PyLT does not complex with p53, unlike the SV40 LT antigen that shares many other biochemical properties with PyLT, including pRb binding. Py has apparently evolved an alternative strategy to inhibit p53 function. This observation strengthens the notion that inactivation of p53 is essential for the propagation of small DNA tumor viruses.

Our results raise the question as to the molecular mechanism(s) by which PymT and Pyst antagonize p53. We did not observe any effect of PymT or Pyst on p53-dependent transactivation of two important p53 target genes, p21 and Bax, indicating that PymT and Pyst act further downstream in the p53 pathway. PymT is associated with cellular membranes and functions as a membrane receptor (reviewed in Ref. 39 ). It has no enzymatic activity itself but binds and alters the function of several host proteins, including PP2A (40 , 41) , the three Src-related kinases c-Src, c-Yes, and Fyn (42) , SHC (43 , 44) , the Mr 85,000 regulatory component of PI3 kinase (35) , some members of the 14-3-3 family of proteins (45) , and phospholipase C-{gamma}1 (46) . Growth factor activation of a signaling pathway from PI3 kinase to the serine/threonine PKB/Akt can deliver a survival signal (32 , 33 , 47) . Such signaling was shown to stimulate inactivation of the death-promoting BAD protein by phosphorylation (48) , providing a direct molecular link between growth factor stimulation and cell survival. Upon phosphorylation at Tyr-315, PymT can bind to the two Src homology 2 domains in the Mr 85,000 component of PI3 kinase and thereby activate PI3 kinase function (39) , followed by PKB/Akt activation (49 , 50) . Thus, it is conceivable that PymT promotes cell survival and antagonizes p53-induced apoptosis through the PI3 kinase/Akt pathway. This idea was supported by our experiments with wortmannin, a PI3 kinase-specific inhibitor that binds to the Mr 110,000 subunit of PI3 kinase. The ability of PymT alone or PymT and Pyst in cooperation to protect cells from p53-induced apoptosis was substantially reduced in the presence of this inhibitor (Fig. 11)Citation . These results are in agreement with the observation that constitutively active PI3 kinase and PKB/Akt can inhibit p53-induced apoptosis (51) . The fact that wortmannin treatment did not affect the partial Pyst-mediated protection from p53-induced apoptosis indicates that Pyst acts independently or downstream of PI3 kinase.

PymT-mediated PI3 kinase activation was shown to block apoptosis induced by serum withdrawal (52) . However, PymT+st did not protect our cells from apoptosis induced by growth in medium containing 0.1% serum. Clearly, coexpression of PymT and Pyst does not endow these cells with a general resistance to apoptosis. Because we have not tested the ability of PymT+st coexpression to inhibit apoptosis induced by a wide range of agents, we do not know whether their antiapoptotic effect is specific for p53-induced apoptosis. Yet it appears likely that PymT and Pyst might also counteract some forms of p53-independent apoptosis, given that they do not interact physically with p53 but rather exert their effect downstream of p53.

The mechanisms behind the cooperativity of Pyst and PymT in the inhibition of p53-induced cell death remain unclear. Our results are consistent with the idea that Pyst provides a PI3 kinase-independent protection against p53-induced apoptosis. This effect of Pyst may give full apoptosis protection only in combination with PymT-mediated activation of the PI3 kinase pathway. The SV40 st antigen and both PymT and Pyst have been shown to bind PP2A (40 , 41) . PP2A is a negative regulator of PKC{zeta}, an nuclear factor-{kappa}B-activating kinase that is involved in mitogenic and survival signaling (53) . One attractive hypothesis is that Pyst binding to PP2A prevents PP2-mediated inhibition of PKC{zeta} activity, leading to nuclear factor-{kappa}B gene activation and increased survival. Because PKC{zeta} can be stimulated in vitro by PI3 kinase, coexpression of PymT that activates PI3 kinase and Pyst that stimulates PKC{zeta} may cooperatively enhance cell survival and thus counteract p53-induced apoptosis. In addition, Pyst has been shown to stimulate cell cycle progression and activate the c-fos promoter through its interaction with PP2A (54) . This could explain the need of both PymT and Pyst for inhibition of cell cycle arrest induced by p53.

In our hands, p53-induced cell cycle arrest was only partially inhibited by PymT and Pyst, whereas p53-induced apoptosis was almost completely blocked. PyLT, which we have not tested in our system, has been shown to interfere with p53-induced cell cycle arrest through its ability to bind and inactivate pRb (55) . We would thus predict that coexpression of the full set of polyoma T antigens (LT, mT, and st) would result in a complete block of p53-induced cell cycle arrest and apoptosis.

Materials and Methods

Cells and Cell Culture.
J3D-M6H4, here denoted M6H4, is a clone of the v-myc retrovirus-induced, p53-negative mouse T-lymphoma line transfected with the ts Val-135 mutant mouse p53 construct (26) . Cells were grown in Iscove’s medium supplemented with 10% FCS at 37°C or 32°C. The incubator temperature was monitored with a mercury thermometer placed in a water-containing tissue culture flask.

Plasmid Construction and Transfection.
To generate the PymT expression vector, a 1.3-kb XhoI-EcoRl fragment containing the entire open reading frame of PymT was isolated from the pneoMLVmT plasmid (56) and cloned into the unique SnaBl-EcoRl sites in the pBabe-puro vector (57) . For the pBabe-Pyst expression vector, a 2.1-Kb BamHI-EcoRI fragment only containing the entire open reading frame of Pyst was isolated from the pAT153/ST1 plasmid (a gift from Göran Magnusson, Uppsala University, Uppsala, Sweden) and cloned into the unique BamHI-EcoRI sites in the pBabe-puro vector. For the pBabe-PymT+st expression vector, a 2.1-kb BamHI-EcoRI fragment containing the entire open reading frame of PymT and Pyst was isolated from the pbc1051 plasmid (58) and cloned into the unique BamHl-EcoRl sites in the pBabe-puro vector. M6H4 cells were stably transfected with the pBabe-PymT, pBabe-st, or pBabe-PymT+st expression vectors by electroporation as described (26) . The presence of pBabe-PymT, pBabe-Pyst, or pBabe-PymT+st in individual clones was confirmed by PCR using the PymT-specific primers 5'-ATGGATAGAGTTCTGAGCAGAG-3' and 5'-CTAGAAATGCCGGGAACG-3' or the Pyst-specific primers 5'-ATGGATAGAGTTCTGAGCAGAG-3' and 5'-CGTGTAGTGGACTGTGGC-3'. PCR was performed by denaturing the DNA at 94°C for 2 min, followed by 25 cycles of amplification at 94°C for 20 s, 54°C for 30 s, 72°C for 1 min and 30 s, and a final extension step at 72°C for 5 min on a Perkin-Elmer GeneAmp PCR system 9600. The empty vector pBabe-puro carrying the puromycin resistance gene was transfected into M6H4 cells as control. The expression of PymT and Pyst was confirmed by Western blotting.

Western Blot Analysis.
Total cell extracts were prepared by lysis in lysis buffer [100 mM Tris (pH 8.0), 150 mM NaCl, 1% NP40, 1 mM phenylmethylsulfonyl fluoride]. Protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Samples containing 50 µg of protein were separated on 8–12% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Blocking and incubation with antibodies were performed in 5% milk in PBS for p53 and PymT detection. For detection of Pyst, 0.1% Tween 20 was added to the blocking solution, and incubation with antibody was performed in 1% milk with 0.1% Tween 20 in PBS. Immunodetection using the ECL system (Amersham Pharmacia Biotech) was carried out according to the manufacturer’s instructions. The rat monoclonal antibody 815 against PymT antigen was provided by Thomas Benjamin (Harvard Medical School, Boston, MA), and the mouse monoclonal antibody F4 against all three polyomavirus T antigens was provided by Göran Magnusson (Uppsala University). p53 was detected with the mouse monoclonal antibody PAb122 (PharMingen, San Diego, CA).

Analysis of Cell Cycle Distribution.
Cells were centrifuged and resuspended in 0.5 ml of a solution containing 50 µg/ml propidium iodide (Sigma Chemical Co., St. Louis, MO), 0.6% NP40, and 0.1% sodium citrate. The stained cells were analyzed in a FACS (Becton Dickinson, CA). The percentage of cells in different phases of the cell cycle was determined by using the CELLFIT and SOBR programs.

Detection of Apoptosis.
Detection of apoptosis by propidium iodide and FACS was performed as described (28 , 59) . Cells were stained with 50 µg/ml propidium iodide in PBS containing 10% FCS. A total of 1 x 104 cells were processed for FACS analysis. The percentage of dead cells was determined by using the LYSIS ll program. The X axis represents relative fluorescence intensity, which is proportional to plasma membrane permeability (the cellular uptake of propidium iodide).

Northern Blot Analysis.
RNA was prepared, and Northern blotting was performed as described (60) . Briefly, 10 µg of total RNA from each sample were fractionated on formaldehyde-agarose gels, transferred to nylon filters, and hybridized with a mouse p21 or Bax cDNA probe. The integrity of the RNA and loading differences were assessed by hybridization with a human glyceraldehyde-3-phosphate dehydrogenase cDNA probe.

Acknowledgments

We thank Thomas L. Benjamin, Harvard Medical School, Boston, MA, for the plasmid pneoMLVmT containing PymT cDNA and the anti-PymT monoclonal antibody 815; Göran Magnusson, Uppsala University, Uppsala, Sweden, for the plasmid pAT153/ST1 and the monoclonal antibody F4 against all three Py T antigens; Stig Linder, Karolinska Institute, for the plasmid pbc1051 containing PymT and Pyst cDNA; Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD, for the mouse WAF1/p21 plasmid; Stanley J. Korsmeyer, Dana-Farber Cancer Institute, Boston, MA, for the mouse Bax plasmid; and Ismail Okan, Karolinska Institute, for subcloning M6H4 from J3D-M6 cells.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Swedish Cancer Society, Magnus Bergvalls Stiftelse, Åke Wibergs Stiftelse, Robert Lundbergs Minnesstiftelse, and Svenska Sällskapet för Medicinsk Forskning. Back

2 To whom requests for reprints should be addressed. Phone: 46-8-728-67-35; Fax: 46-8-33-04-98; E-mail: Klas.Wiman{at}mtc.ki.se Back

3 The abbreviations used are: pRb, retinoblastoma protein; Py, polyoma virus; LT, large T; mT, middle T; st, small t; ts, temperature sensitive; FACS, fluorescence-activated cell sorter; PI3 kinase, phosphatidylinositol 3-kinase; PP2A, protein phosphatase 2A; PK, protein kinase. Back

Received for publication 5/14/99. Revision received 11/ 4/99. Accepted for publication 12/ 1/99.

References

  1. Greenblatt M. S., Bennett W. P., Hollstein M., Harris C. C. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res., 54: 4855-4878, 1994.[Free Full Text]
  2. Donehower L. A., Harvey M., Slagle B. L., McArthur M. J., Montgomery C. A., Jr., Butel J. S., Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors. Nature (Lond.), 356: 215-221, 1992.[Medline]
  3. Ko L. J., Prives C. p53: puzzle and paradigm. Genes Dev., 10: 1054-1072, 1996.[Free Full Text]
  4. Sherr C. J. Tumor surveillance via the ARF-p53 pathway. Genes Dev., 12: 2984-2991, 1998.[Free Full Text]
  5. Symonds H., Krall L., Remington L., Saenz-Robles M., Lowe S., Jacks R., Van Dyke T. p53-dependent apoptosis suppresses tumor growth and progression in vivo. Cell, 78: 703-711, 1994.[Medline]
  6. El-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell, 75: 817-825, 1993.[Medline]
  7. El-Deiry W. S., Harper J. W., O’Connor P. M., Velculescu V. E., Canman C. E., Jackman J., Pietenpol J. A., Burrell M., Hill D. E., Wang Y., Wiman K. G., Mercer W. E., Kastan M. B., Kohn K. W., Elledge S. J., Kinzler K. W., Vogelstein B. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res., 54: 1169-1174, 1994.[Abstract/Free Full Text]
  8. Kastan M. B., Zhan Q., El-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell, 71: 587-597, 1992.[Medline]
  9. Hermeking H., Lengauer C., Polyak K., He T. C., Zhang L., Thiagalingam S., Kinzler K. W., Vogelstein B. 14-3-3 {varsigma} is a p53-regulated inhibitor of G2/M progression. Mol. Cell, 1: 3-11, 1997.[Medline]
  10. Utrera R., Collavin L., Lazarevic D., Delia D., Schneider C. A novel p53-inducible gene coding for a microtubule-localized protein with G2-phase-specific expression. EMBO J., 17: 5015-5025, 1998.[Medline]
  11. Miyashita T., Reed J. C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell, 80: 293-299, 1995.[Medline]
  12. Owen-Scaub L. B., Zhang W., Cusack J. C., Angelo L. S., Santee S. M., Fujiwara T., Roth J. A., Deisseroth A. B., Zhang W. W., Kruzel E., Radinsky R. Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression. Mol. Cell. Biol., 15: 3032-3040, 1995.[Abstract/Free Full Text]
  13. Wu G. S., Burns T. F., McDonald E. R., III, Jiang W., Meng R., Krantz I. D., Kao G., Gan D. D., Zhou J. Y., Muschel R., Hamilton S. R., Spinner N. B., Markowitz S., Wu G., El-Deiry W. S. KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat. Genet., 17: 141-143, 1997.[Medline]
  14. Buckbinder L., Talbott R., Velasco-Miguel S., Takenaka I., Faha B., Seizinger B. R., Kley N. Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature (Lond.), 377: 646-649, 1995.[Medline]
  15. Polyak K., Xia Y., Zweier J. L., Kinzler K. W., Vogelstein B. A model for p53-induced apoptosis. Nature (Lond.), 389: 300-305, 1997.[Medline]
  16. El-Deiry W. S. Regulation of p53 downstream genes. Semin. Cancer Biol., 8: 345-357, 1998.[Medline]
  17. Gottlieb T. M., Oren M. p53 and apoptosis. Semin. Cancer Biol., 8: 359-368, 1998.[Medline]
  18. Selivanova G., Wiman K. G. p53. A cell cycle regulator activated by DNA damage. Adv. Cancer Res., 66: 143-180, 1995.[Medline]
  19. Teodoro J. G., Branton P. E. Regulation of apoptosis by viral gene products. J. Virol., 71: 1739-1746, 1997.[Free Full Text]
  20. Weinberg R. A. The retinoblastoma protein and cell cycle control. Cell, 81: 323-330, 1995.[Medline]
  21. Topp W. C., Lane D. P., Poolack R. Transformation by simian virus 40 and polyoma virus Tooze J. eds. . DNA Tumor Viruses: Molecular Biology of Tumor Viruses, : 205-296, Cold Spring Harbor Laboratory Cold Spring Harbor, NY 1981.
  22. Acheson N. H. Lytic cycle of SV40 and polyoma virus Tooze J. eds. . DNA Tumor Viruses: Molecular Biology of Tumor Viruses, : 61-124, Cold Spring Harbor Laboratory Cold Spring Harbor, NY 1981.
  23. Soeda E., Arrand J. R., Smolar N., Walsh J. E., Griffin B. E. Coding potential and regulatory signals of the polyoma virus genome. Nature (Lond.), 283: 445-453, 1980.[Medline]
  24. Turler H. The tumor antigens and the early functions of polyoma virus. Mol. Cell. Biol., 32: 63-93, 1980.
  25. Wang E. H., Friedman P. N., Prives C. The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen. Cell, 57: 379-792, 1989.[Medline]
  26. Wang Y., Szekely L., Okan I., Klein G., Wiman K. G. Wild type p53-triggered apoptosis is inhibited by bcl-2 in a v-myc-induced T-cell lymphoma line. Oncogene, 8: 3427-3431, 1993.[Medline]
  27. Wang Y., Okan I., Szekely L., Klein G., Wiman K. G. Bcl-2 inhibits wild type p53-triggered apoptosis but not G1 cell cycle arrest and transactivation of WAF1 and bax. Cell Growth Differ., 6: 1071-1075, 1995.[Abstract]
  28. Haas-Kogan D. A., Kogan S. C., Levi D., Dazin P., Táng A., Fung Y. K. T., Israel M. A. Inhibition of apoptosis by the retinoblastoma gene product. EMBO J., 14: 461-472, 1995.[Medline]
  29. Treisman R., Noval U., Favaloro J., Kamen R. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature (Lond.), 292: 595-600, 1981.[Medline]
  30. Asselin C., Gelinas C., Bastin M. Role of the three polyomavirus early proteins in tumorigenesis. Mol. Cell. Biol., 3: 1451-1459, 1983.[Abstract/Free Full Text]
  31. Asselin C., Vass-Marego J., Bastin M. Mutation in the polyomavirus genome that activates the properties of large T associated with neoplastic transformation. J. Virol., 57: 165-172, 1986.[Abstract/Free Full Text]
  32. Franke T. F., Kalpan D. R., Cantley L. C. PI3K: downstream AKTion blocks apoptosis. Cell, 88: 435-437, 1997.[Medline]
  33. Kennedy S. G., Wagner A. J., Conzen S. D., Jordan J., Bellacosa A., Tsichlis P. N., Hay N. The PI 3-kinase/AKt signaling pathway delivers an anti-apoptotic signal. Genes Dev., 11: 701-713, 1997.[Abstract/Free Full Text]
  34. Whitman M., Kaplan D. R., Schaffhausen B. S., Cantley L., Roberts T. M. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature (Lond.), 315: 239-242, 1985.[Medline]
  35. Kaplan D. R., Pallas D. C., Morgan W., Schaffhausen B., Roberts T. M. Mechanisms of transformation by polyoma virus middle T antigen. Biochim. Biophys. Acta, 948: 345-364, 1988.
  36. Talmage D. A., Freund R., Young A. T., Dahl J., Dawe C. J., Benjamin T. Phosphorylation of middle T by pp60c-src: a switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis. Cell, 59: 55-65, 1989.[Medline]
  37. Serunian L. A., Auger K. R., Roberts T. M., Cantley L. C. Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen. J. Virol., 64: 4718-4725, 1990.[Abstract/Free Full Text]
  38. Yano H., Nakanishi S., Kimura K., Hanai N., Saitoh Y., Fukui Y., Nonomura Y., Matsuda Y. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J. Biol. Chem., 268: 25846-25856, 1993.[Abstract/Free Full Text]
  39. Dilworth S. M. Polyoma virus middle T antigen: meddler or mimic?. Trends Microbiol., 3: 31-35, 1995.[Medline]
  40. Pallas D. C., Shahrik L. K., Martin B. L., Jaspers S., Miller T. B., Brautigan D. L., Roberts T. M. Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell, 60: 167-176, 1990.[Medline]
  41. Walter G., Ruediger R., Slaughter C., Mumby M. Association of protein phosphatase 2A with polyoma virus medium tumor antigen. Proc. Natl. Acad. Sci. USA, 87: 2521-2525, 1990.[Abstract/Free Full Text]
  42. Messerschmitt A. S., Dunant N., Ballmer-Hofer K. DNA tumor viruses and Src family tyrosine kinase, an intimate relationship. Virology, 227: 271-280, 1997.[Medline]
  43. Campbell K. S., Ogris E., Burke B., Su W., Auger K. R., Druker B. J., Schaffhausen B. S., Roberts T. M., Pallas D. C. Polyoma middle T antigen interacts with SHC via the NPTY motif in middle T. Proc. Natl. Acad. Sci. USA, 91: 6344-6348, 1994.[Abstract/Free Full Text]
  44. Dilworth S. M., Brewster C. E., Jone M. D., Lanfrancone L., Pelicci G., Pelicci P. G. Transformation by polyoma virus middle T-antigen involves the binding and tyrosine phosphorylation of Shc. Nature (Lond.), 367: 87-90, 1994.[Medline]
  45. Pallas D. C., Fu L., Haehnel L. C., Weller W., Collier R. J., Roberts T. M. Association of polyomavirus middle tumor antigen with 14-3-3 protein. Science (Washington DC), 265: 535-537, 1994.[Abstract/Free Full Text]
  46. Su W., Liu W., Schaffhausen B. S., Roberts T. M. Association of polyomavirus middle tumor antigen with phospholipase C-{gamma}1. J. Biol. Chem., 270: 12331-12334, 1995.[Abstract/Free Full Text]
  47. Eves E. M., Xiong W., Bellacosa A., Kennedy S. G., Tsichlis P. N., Rosner M. R., Hay N. Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line. Mol. Cell. Biol., 18: 2143-2152, 1998.[Abstract/Free Full Text]
  48. Datta S. R., Dudek H., Tao X., Masters S., Fu H., Gotoh Y., Greenberg M. E. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell, 91: 231-241, 1997.[Medline]
  49. Meili R., Cron P., Hemmings B. A., Ballmer-Hofer K. Protein kinase B/Akt is activated by polyomavirus middle-T antigen via a phosphatidylinositol 3-kinase-dependent mechanism. Oncogene, 16: 903-907, 1998.[Medline]
  50. Summer S. A., Lipfert L., Birnbaum M. J. Polyoma middle T antigen activates the Ser/Thr kinase Akt in a PI3-kinase-dependent manner. Biochem. Biophys. Res. Commun., 246: 76-81, 1998.[Medline]
  51. Sabbatini P., McCormick F. Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis. J. Biol. Chem., 274: 24263-24269, 1999.[Abstract/Free Full Text]
  52. Dahl J., Jurczak A., Cheng L. A., Baker D. C., Benjamin T. L. Evidence of a role for phosphatidylinositol 3-kinase activation in the blocking of apoptosis by polyomavirus middle T antigen. J. Virol., 72: 3221-3226, 1998.[Abstract/Free Full Text]
  53. Sontag E., Sontag J. M., Garcia A. Protein phosphatase 2A is a critical regulator of protein kinase C {zeta} signaling targeted by SV40 small t to promote cell growth and NF-kB activation. EMBO J., 16: 5662-5671, 1997.[Medline]
  54. Mullane K. P., Ratnofsky M., Cullere X., Schaffhausen B. Signaling from polyomavirus middle T and small T defines different roles for protein phosphatase 2A. Mol. Cell. Biol., 18: 7556-7564, 1998.[Abstract/Free Full Text]
  55. Doherty J., Freund R. Polyomavirus large T antigen overcomes p53 dependent growth arrest. Oncogene, 14: 1923-1931, 1997.[Medline]
  56. Dahl J., Thathamangalam U., Freund R., Benjamin T. L. Functional asymmetry of the regions juxtaposed to the membrane binding sequence of polyomavirus middle T antigen. Mol. Cell. Biol., 12: 5050-5058, 1992.[Abstract/Free Full Text]
  57. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res., 18: 3587-3596, 1990.[Abstract/Free Full Text]
  58. Linder S., Nilsson M., Martens I., Magnusson G. A viable mouse polyomavirus mutant without immortalizing or transforming activities. Virology, 179: 78-86, 1990.[Medline]
  59. Wang Y., Okan I., Pokrovskaja K., Wiman K. G. Abrogation of p53-induced G1 arrest by the HPV 16 E7 protein does not inhibit p53-induced apoptosis. Oncogene, 12: 2731-2735, 1996.[Medline]
  60. Qian W., Kashuba E., Magnusson K. P., Pokrovskaja K., Okan I., Klein G., Wiman K. G. Role of p53 mutation in polyomavirus-induced tumorigenesis. J. Gen. Viol., 78: 893-903, 1997.[Abstract]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Andrabi, O. V. Gjoerup, J. A. Kean, T. M. Roberts, and B. Schaffhausen
Protein phosphatase 2A regulates life and death decisions via Akt in a context-dependent manner
PNAS, November 27, 2007; 104(48): 19011 - 19016.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
N. W. Plummer, C. J. Gallione, S. Srinivasan, J. S. Zawistowski, D. N. Louis, and D. A. Marchuk
Loss of p53 Sensitizes Mice with a Mutation in Ccm1 (KRIT1) to Development of Cerebral Vascular Malformations
Am. J. Pathol., November 1, 2004; 165(5): 1509 - 1518.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
D. Dey, J. Dahl, S. Cho, and T. L. Benjamin
Induction and Bypass of p53 during Productive Infection by Polyomavirus
J. Virol., September 15, 2002; 76(18): 9526 - 9532.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
K. A. Gottlieb and L. P. Villarreal
Natural Biology of Polyomavirus Middle T Antigen
Microbiol. Mol. Biol. Rev., June 1, 2001; 65(2): 288 - 318.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
A. Yen, L. Placanica, S. Bloom, and S. Varvayanis
Polyomavirus Small t Antigen Prevents Retinoic Acid-Induced Retinoblastoma Protein Hypophosphorylation and Redirects Retinoic Acid-Induced G0 Arrest and Differentiation to Apoptosis
J. Virol., June 1, 2001; 75(11): 5302 - 5314.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qian, W.
Right arrow Articles by Wiman, K. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qian, W.
Right arrow Articles by Wiman, K. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation